[show abstract][hide abstract] ABSTRACT: This paper presents an optimized procedure for assessing an immune-mediated cytotoxicity, produced after the addition of human and baboon serum to transgenic porcine fibroblasts. This procedure is performed with the xCELLigence Real-Time Cell Analyzer (RTCA). The xCELLigence system measures the impedance variations in the culture media of a 96-well microelectronic plate, and shows the changes in cell number and morphology in a real-time plot. However, different factors need to be optimized before developing an RTCA assay. Thus, we studied the influence of several variables, such as the number of cells seeded, the time the cells were allowed to grow before the tests, the serum concentration and the addition of rabbit complement. The findings were confirmed by the WST-1 classical cytotoxicity test. The results showed that 7.5 × 10(3) cells seeded per well produced the adequate CI in 10 h. The area under the curve and the CImin versus concentration values showed a very high correlation index (r(2) = 0.966 and r(2) = 0.92 for the first 50 h after challenge, respectively), proving that CI variations are directly proportional to the quantity of serum added. The addition of complement resulted in lower CImin values. Therefore, both the cytolysis level with and without exogenous complement addition had to be assessed. There was a high correlation between the relative cytotoxicity assessed by WST-1 and the CI obtained by RTCA when exogenous complement was not added (r(2) = 0.827; p < 0.001). The correlation was average when rabbit complement was added (r(2) = 0.523; p = 0.046). In conclusion, culture conditions have an important influence on RTCA cytotoxicity assays.
[show abstract][hide abstract] ABSTRACT: A 12-year-old, male, fox terrier dog presented with an abnormal gait of the left pelvic limb. Computed tomography revealed a large, homogeneous, hypoattenuating, noncontrast enhancing mass within the left epaxial muscles that invaded the L5-6 vertebral canal and caused spinal cord compression. Imaging findings were consistent with an infiltrative lipoma. The mass was removed and a left hemilaminectomy was performed in the affected area. Histopathology confirmed the mass to be an infiltrative lipoma. The dog recovered and regained neurologic function within 2 weeks. Computed tomography assisted preoperative planning by characterizing the shape, size, and location of the mass.
[show abstract][hide abstract] ABSTRACT: Objective-To characterize the kinetics of interleukin (IL)-4, IL-5, and IL-13 secretion in peripheral blood and lymph node mononuclear cells isolated from porcine circovirus type 2 (PCV2)-vaccinated pigs after cells were challenged with PCV2 open reading frame 2 antigen. Animals-10 pigs. Procedures-5 pigs were vaccinated with a PCV2 vaccine and received a booster dose 3 weeks later. They were kept together with a similar group of 5 nonvaccinated pigs that served as controls. One week after the second vaccination, peripheral blood mononuclear cells (PBMCs) and excised retropharyngeal lymph node mononuclear cells (LNMCs) were isolated and cultured. Cells were then challenged by exposure to PCV2 open reading frame 2 and evaluated at 2, 12, 24, and 48 hours to determine the expression of IL-4, IL-5, and IL-13 via quantitative PCR assay. Changes in gene expression were analyzed relative to the results from analysis of the sample at 0 hours (calibrator). Results-All ILs were upregulated differently in LNMCs and PBMCs from vaccinated pigs. Lymph node mononuclear cells from vaccinated animals produced significantly more IL-4 mRNA than did PBMCs at 2, 12, and 48 hours (relative change: 2.8 vs -3.6, 13.0 vs 3.6, and 9.8 vs 1.8, respectively) and more IL-5 mRNA at 2, 12, 24, and 48 hours (relative change: 1. 2 vs -4.8, 2.2 vs 0.2, 3.2 vs -1.9, and 4.0 vs -3.6, respectively). Interleukin-13 mRNA reached its highest concentration at 24 hours but was 11.9-fold higher in PBMCs than in LNMCs. Conclusions and Clinical Relevance-Results supported the importance of IL-4, IL-5, and IL-13 in pigs, suggesting that PBMCs and LNMCs express cytokines in a tissue-specific manner.
American Journal of Veterinary Research 01/2013; 74(1):110-4. · 1.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) impairs local pulmonary immune responses by damaging the mucociliary transport system, impairing the function of porcine alveolar macrophages andinducing apoptosis of immune cells. An imbalance between pro- and anti-inflammatory cytokines, including tumour necrosis factor-α and interleukin-10, in PRRS may impair the immune response of the lung. Pulmonary macrophage subpopulations have a range of susceptibilities to different PRRSV strains and different capacities to express cytokines. Infection with PRRSV decreases the bactericidal activity of macrophages, which increases susceptibility to secondary bacterial infections. PRRSV infection is associated with an increase in concentrations of haptoglobin, which may interact with the virus receptor (CD163) and induce the synthesis of anti-inflammatory mediators. The balance between pro- and anti-inflammatory cytokines modulates the expression of CD163, which may affect the pathogenicity and replication of the virus in different tissues. With the emergence of highly pathogenic PRRSV, there is a need for more information on the immunopathogenesis of different strains of PRRS, particularly to develop more effective vaccines.
The Veterinary Journal 12/2012; · 2.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Metabolic and cardiovascular diseases (CVDs) have risen to alarming proportions, and there is a need for therapeutic and preventive measures. The polyphenol resveratrol (RES) protects against CVDs, but in vivo molecular mechanisms responsible for protection are not yet understood. Peripheral blood mononuclear cells (PBMNCs) are involved in the development of atherosclerosis and metabolic disorders. The identification of PBMNCs genes responding to dietary compounds might help to understand the mechanisms underlying the effects of polyphenols. We determined gene expression differences between PBMNCs from pigs fed a high-fat diet manifesting a mild increase of cholesterol and pigs fed a high-fat diet containing low doses of RES. Although the consumption of RES did not modify the levels of cholesterol, microarray analyses indicated that some of the differentially expressed genes, collagens (COL1A, COL3A), lipoprotein lipase (LPL) and fatty-acid binding proteins (FABPs) involved in CVDs and lipid metabolism were up-regulated by the high-fat diet and down-regulated by RES. Reverse transcriptase polymerase chain reaction confirmed that RES and RES-containing grape extract prevented the induction of FABP4 in PBMNCs in female pigs fed a high-fat diet. Low micromolar concentrations of RES and its metabolite dihydroresveratrol exerted a minor but significant reducing effect on the induction of FABP4 expression in human macrophages treated with oxidized low-density lipoprotein. Our results show that the consumption of low doses of RES modulates the expression of genes related to lipid metabolism and metabolic disorders that are affected by a high-fat diet and suggest that some of the circulating RES metabolites may contribute to these effects.
The Journal of nutritional biochemistry. 08/2012; 23(7):829-37.
[show abstract][hide abstract] ABSTRACT: Despite the numerous studies carried out, the mechanisms used by porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) to impair the host immune response are not yet clear. The aim of this study was to determine the expression of IL-12, IL-10, IFN-α and IFN-γ in lymphoid organs of PRRSV experimentally-infected pigs. Twenty eight piglets were inoculated with PRRSV field isolate 2982 and killed in batches of four at 3, 7, 10, 14, 17, 21 and 24 days post-inoculation (dpi). Control animals were mock-inoculated and killed at the end of the study. Samples from mediastinal and retropharyngeal lymph nodes and tonsil were collected and fixed for histopathological and immunohistochemical analyses. PRRSV antigen was mainly detected in the cytoplasm of macrophages, displaying a bimodal expression with a first peak at 3-7dpi and a second peak at 14dpi. The expression of IFN-α showed an early enhancement at 3dpi, and both IL-12 and IFN-γ displayed a similar trend in all the lymphoid organs analysed, showing an increase at 3-7dpi and at 14-17dpi. On the other hand, the expression of IL-10 was lower than the one observed for the other cytokines. The expression of IL-10 compared with the higher expression of IL-12, IFN-α and IFN-γ detected in this study, indicates that other mechanisms besides the expression of IL-10 play a role in the inducement of an erratic host immune response. Taking into account the enhanced expression of IFNs together with the detection of PRRSV antigen until the end of the study in the examined lymphoid organs, further studies are being conducted to rule out a down-regulation in IFN signalling pathway.
Veterinary Immunology and Immunopathology 07/2012; 149(3-4):262-71. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) can persist in different organs of infected pigs, which suggests a failure in the immune response. Antigen-presenting cells (APCs) play a pivotal role in the induction of effective T- and B-cell responses. In this study, we investigated the changes in the different APC subpopulations and T- and B-cell counts in the tonsil, retropharyngeal and mediastinal lymph nodes of pigs experimentally infected with a European PRRSV field isolate. Our results demonstrated that the expression of S100, SWC3, HLA-DR molecule and CD3 was diminished in the studied organs throughout the study, observing a significant negative correlation between viral antigen and HLA-DR expression in both retropharyngeal and mediastinal lymph nodes. In contrast, λ-light chains showed an increase during the study. Taking all into account, after PRRSV infection, no enhancement in the number of APCs and T cells was observed, suggesting an impairment of the immune function which may allow the persistence of PRRSV into the organism.
Transboundary and Emerging Diseases 07/2012; · 2.10 Impact Factor
[show abstract][hide abstract] ABSTRACT: Using a percutaneous ecoguided injection system to obtain chimeric piglets through a less invasive and traumatic technique than previously reported.
The two types of human cells included umbilical cord blood mononuclear elements and mesenchymal stem cells cultured from bone marrow. Four sows at gestational day 50 were anesthetized. A needle was inserted through the skin and uterine wall to reach the peritoneal cavity of the fetuses under continuous ultrasound guidance. Fourteen piglets were injected with various cell concentrations.
All sows carried pregnancies to term yielding 69 piglets, among which 67 were alive and two mummified. Two piglets died during the first 48 hours of life. Chimerism was detected using flow cytometry and by quantitative polymerase chain reaction (q-PCR) to detect Alu gene in blood or tissues samples. The analysis detected blood chimerism in 13 piglets (21%) by flow cytometry and the presence of the human Alu gene in 33 (51%) by q-PCR. The results suggest cell trafficking between littermates after in utero injection.
Transcutaneous echo-guided injection succeeded to produce chimeric piglets without disadvantages to the sow or the fetuses and avoiding abortions or fetal death.
[show abstract][hide abstract] ABSTRACT: The presence of grape and wine polyphenol resveratrol (RES) in the diet is negligible. Therefore, the cardiovascular benefits of this molecule, in a dietary context, remain to be established. We aimed to investigate, through dietary intervention, the effects of a resveratrol-rich grape extract (GE-RES) on the prevention of early aortic lesions in pigs fed an atherogenic diet (AD). These effects were compared with those produced by a grape extract lacking RES (GE) or RES alone. Pigs fed the AD for 4 months showed early atherosclerotic lesions in the thoracic aorta: degeneration and fragmentation of elastic fibers, increase of intima thickness, subendothelial fibrosis, and accumulation of fatty cells and anion superoxide radicals. GE-RES was the most effective treatment and prevented the disruption of aortic elastic fibers, decreased their alteration (57%), and reduced the intima thickness (33%) and the accumulation of fatty cells (42%) and O(2)(•-) (38%) in aortic tissue. In addition, GE-RES moderately downregulated the expression of the suppressors of cytokine signaling 1 (SOCS1) and 3 (SOCS3), key regulators of vascular cell responses, in peripheral mononuclear blood cells. Our results suggest that the consumption of this GE-RES nutraceutical, in a dietary prevention context, could prevent early atherosclerotic events. The presence of RES in the grape extract strengthened these effects.
Journal of Agricultural and Food Chemistry 05/2012; 60(22):5609-20. · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The risk of zoonoses is a major obstacle to xenotransplantation. Porcine endogenous retrovirus (PERV) poses a potential risk of zoonotic infection, and its control is a prerequisite for the development of clinical xenotransplantation. The copy number of PERV varies among different breeds, and it has been suggested that the PERV integrations number is increased by inbreeding. The purpose of this study was (i) to examine the copy number of PERV in different Spanish pig breeds, Spanish wild boar and commercial cross-bred pigs from five different farms and genetic background (CCP1-CCP5) and (ii) to investigate the correlation between PERV copy number and the genetic background of the pigs in order to improve the selection of pigs for xenotransplantation. PERV copy number was determined by quantitative, real-time polymerase chain reactions. Thirty-four microsatellite markers were genotyped to describe the genetic diversity within populations (observed and expected heterozygosities, Ho and He, respectively) and the inbreeding coefficient (F). Pearson's correlation coefficient was used to determine the relationship between PERV copy number and Ho, He and F. The copy number of PERV among different pig breeds was estimated to range between three (CCP1) and 43 copies (Iberian Pig). Statistical differences were found among the studied populations concerning PERV copy number. No correlation was found between the PERV copy number and the heterozygosity (calculated at an individual level or at a population level) or the inbreeding coefficient of each population. Our data suggest that pigs inbreeding does not increase PERV copy number and support the idea that careful selection of pigs for organ donation with reduced PERV copy number will minimize the risk of retrovirus transmission to the human receptor.
Zoonoses and Public Health 02/2012; 59(6):401-7. · 2.09 Impact Factor
[show abstract][hide abstract] ABSTRACT: Transforming growth factor β (TGFβ) is an immunomodulatory cytokine which is able to modulate the host immune response eliciting an inefficient response against pathogens. In this sense, the role of this cytokine in porcine reproductive and respiratory syndrome (PRRS) has been poorly studied and the reported results are contradictory. Thus, in the present study, the expression of TGFβ was analysed both at tissue (lymphoid organs and lung) and serum level to study its correlation with the expression of PRRS virus (PRRSV). To carry out this study, 32 pigs were inoculated with the European PRRSV field isolate 2982 and sequentially killed from 0 dpi to the end of the study (24 dpi). Blood and tissue samples were collected to determine the expression of PRRSV and TGFβ. PRRSV was detected in inoculated animals from 3 dpi until the end of the study, however TGFβ was not detected in sera from inoculated animals. Contrary, an increase of TGFβ antigen was observed both in the lymphoid organs and in the lung of PRRSV-inoculated pigs when compared with control group. Since TGFβ play a role as an immunomodulatory cytokine of the immune response and also in the differentiation of regulatory T cells (Tregs), the upregulation of the TGFβ at tissue level may play a role in the impairment of the host immune response observed during PRRS, being observed a significant correlation between PRRSV and TGFβ expression at lung level.
[show abstract][hide abstract] ABSTRACT: In order to clarify the origin of the haptoglobin (Hp) quantified in saliva and meat juice samples, the extrahepatic localization of Hp in salivary gland and in diaphragmatic muscle, as part of the systemic acute phase response in pigs, was studied by immunohistochemistry. For this purpose a specific monoclonal antibody (mAb) produced by immunising mice with purified porcine Hp was used. Reactivity of the mAb was assessed by direct ELISA and by western blot, which showed the ability and specificity of the mAb to identify porcine haptoglobin as a purified antigen or in porcine serum in a native or denatured but non-reduced state. Five healthy and five diseased pigs were sampled at slaughter for serum and tissue procurement. Hepatic immunohistochemical analysis was used as control of the acute phase reaction status. In the liver, cell immunostaining revealed a perinuclear, cytoplasmic localization of Hp within hepatocytes, following mainly a periacinar pattern. Extrahepatic immunohistochemical analysis revealed positive cells in the glandular acini and duct epithelial cells of the salivary gland and intrasarcoplasmic immunolabelling of random diaphragmatic myofibers. A possible role of both salivary gland and diaphragmatic muscle on local Hp production could be postulated based on the present immunohistochemical study, which supports the concept that other cells besides hepatocytes may have the potential to produce Hp in the pig.
Histology and histopathology 02/2012; 27(2):187-96. · 2.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The concentrations of haptoglobin (Hp), C-reactive protein (CRP) and serum amyloid A (SAA) were measured in wasted pigs, first to evaluate their usefulness in the diagnosis of infectious, wasting diseases in pigs, and second, to evaluate whether their concentrations can distinguish the lymphoid depletion score in the lymph tissues of wasted affected pigs. Fifty-three wasted pigs and seven specific pathogen free (SPF) pigs were postmortem examined. Gross lesions were evaluated and samples for histopathological, immunohistochemical, molecular biology and microbiological analysis were taken. Thirty-one pigs were diagnosed as postweaning multisystemic wasting syndrome (PMWS) and 22 as porcine respiratory disease complex (PRDC). Lymphoid depletion degree in lymph tissues of PMWS and PRDC affected pigs was determined. Serum Hp was significantly higher in pigs with PRDC in comparison with the PMWS affected pigs. Serum CRP concentration was significantly lower in pigs with PRDC than in PMWS affected pigs (P<0.001). CRP and SAA levels increased with the lymphoid depletion score, presenting statistical differences between pigs with no depletion and pigs with low, moderate or severe lymphoid depletion (P<0.05, P<0.05 and P<0.001 for CRP and P<0.01, P<0.01 and P<0.01 for SAA, respectively). Hp was higher in pigs with no or low depletion compared with the pigs suffering severe lymphoid depletion (P<0.001 and P<0.05, respectively).
[show abstract][hide abstract] ABSTRACT: Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most important diseases in the swine industry. Although several studies have been carried out to elucidate the host immune response evoked against PRRS virus (PRRSV), there are several aspects that still remain unclear. The aim of this study was to determine the expression of IL-1α, IL-6 and TNF-α in the lymphoid organs (mediastinal and retropharyngeal lymph nodes and tonsil) of PRRSV-infected pigs and to determine their correlation with the expression of PRRSV antigen. Proinflammatory cytokine expression was different depending on the body compartment examined. Thus, whereas IL-1α and TNF-α were the main cytokines expressed in the mediastinal lymph node, IL-6 was the most highly expressed cytokine in the retropharyngeal lymph node, and no expression of proinflammatory cytokines was observed in the tonsil. These findings may be related to the impairment of the host immune response evoked after PRRSV infection. Therefore, lymphoid organs and proinflammatory cytokines represent an important target of study for clarifying the immunopathogenesis of PRRS.
Transboundary and Emerging Diseases 08/2011; 59(2):145-53. · 2.10 Impact Factor
[show abstract][hide abstract] ABSTRACT: Scope: trans-Resveratrol (RES) and/(or) its metabolites exert many effects in vivo. Our aim was to study the metabolism and tissue distribution of RES using the pig, a mammal physiologically close to humans.Methods and results: Forty-seven tissues, organs and fluids were analyzed 6 h after intragastric RES administration (5.9 mg/kg body weight) using HPLC-MS/MS. Twelve RES and seven dihydroresveratrol (DH-RES) metabolites were detected. DH-RES was the main metabolite in cecum, colon and rectum, whereas RES-3-O-glucuronide was the most abundant one in fluids and organs. Approximately 74.5% of the total RES administered was recovered in the form of RES, DH-RES and derived metabolites (65.1% along the gastrointestinal tract, 7.7% in urine, 1.2% in bile and 0.5% in organs). We report here, for the first time, the occurrence of RES ribosyl-sulfate derivative, DH-RES diglucuronide, DH-RES sulfoglucuronide and DH-RES disulfate as well as the metabolic profile of RES and DH-RES in the aorta, lymph, lymph node, ovaries, uterus, cerebellum, pancreas, urinary bladder tissue, fat and muscle.Conclusion: This study contributes to the clarification of the metabolism and tissue distribution of RES and could help to further understand the mechanisms underlying its effects.
[show abstract][hide abstract] ABSTRACT: Apoptosis is a strictly regulated mechanism of cell death that involves a complex network of biochemical pathways. Whether a cell undergoes apoptosis or not depends on a delicate balance of anti- and pro-apoptotic stimuli. This phenomenon can be induced by two different pathways: intrinsic and extrinsic pathways. The main aim of this study was to determine the ideal fixative and antigen retrieval method in porcine paraffin embedded tissues for the immunohistochemical detection of apoptosis mediators, from both extrinsic and intrinsic pathways. Tonsil, retropharyngeal lymph node and lung tissue samples were fixed in 10% neutral buffered formalin, Bouin solution and zinc salts fixative (ZSF) and different unmasking methods were carried out. Both 10% neutral buffered formalin and ZSF resulted as the fixatives of election to study apoptosis phenomena. Tween 20 (0.01% in PBS), citrate buffer (microwave, pH 6.0) and/or protease type XIV were the antigen retrieval methods which displayed better labelling. Our results allow to deep in the knowledge of apoptosis and its role in the pathogenesis of porcine diseases.
Veterinary Immunology and Immunopathology 10/2010; 139(2-4):210-6. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: A number of pharmacokinetic studies have shown marked differences in the plasma metabolic profile of resveratrol (RES) between humans and animals and between individuals of the same species, which complicates the identification of the putative bioactive metabolites responsible for the beneficial effects of RES. On the basis of the physiological similarity between pigs and humans, the aim of this work was to characterize the metabolic profile and pharmacokinetics of RES in the plasma of pigs and to compare this to values reported in humans. RES (5.9 mg/kg of body weight) was orally administered to pigs. The following metabolites were identified in plasma using HPLC-MS/MS: RES-diglucuronide (1), two isomers of RES-sulfoglucuronide (2, 3), two isomers of RES-glucuronide (4, 5), RES-sulfate (6), and RES. The most abundant metabolites were 2, 5 (identified as resveratrol 3-O-glucuronide), and 6. The t(max) ranged from 0.9 h for compounds 2 and 5 to 2 h for compound 3. The highest C(max) value was 2223 ng/mL (5.5 μM) for metabolite 5, which was 2.6-, 3.3-, and 12-fold higher than that for metabolites 6, 2, and 3, respectively. Peak plasma levels of RES (53 ng/mL; 0.23 μM) were detected 0.5 h after RES ingestion. Apart from the low levels of RES aglycone, the RES metabolic profile in pigs differs from that found in humans. The identification of the actual active RES metabolites is a challenge that requires more complex studies which should take into account many possible influencing factors such as age, gender, and methodological approaches.
Journal of Agricultural and Food Chemistry 10/2010; · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: To design a real-time quantitative polymerase chain reaction (q-PCR) to assess gene expression for hCD55, hCD59, and hCD46 in polytransgenic (PT) pigs used as xenograft donors for orthotopic liver xenotransplantation using a pig-to-baboon model.
Three pairs of primers were designed using PrimerBlast and mRNA of hCD55, hCD59, and hCD46 sequences. Blood samples from five PT pigs (two males and three females) were used to isolated peripheral blood mononuclear cells (PBMCs) by means of Ficoll gradients. After DNAase digestion of isolated mRNA, we synthesized cDNA. Using SYBR-Green chemistry of q-PCR, we constructed a standard curve. Two wild-type (WT) pigs were used as negative controls, and PBMCs from two healthy human volunteers as positive controls. The amplicon length was assessed by means of agarose gel electrophoresis and PCR products, sequenced.
We observed amplification for hCD55, hCD59, and hCD46 in all samples from the five PT pigs except for hCD55 and hCD46 in one male PT pig. Neither the human samples nor the negative controls showed amplification. The expected amplicon length was confirmed; sequencing showed high homology with human mRNA for the three proteins and no match with any known pig sequence.
The q-PCR allowed detection of animals with the highest gene expression for hCD55, hCD59, and hCD46 for xenograft donors in transplantation experiments.